Ibrutinib Plus Venetoclax for First-Line Treatment of Chronic Lymphocytic Leukemia: Primary Analysis Results From the Minimal Residual Disease Cohort of the Randomized Phase II CAPTIVATE Study.

PURPOSE: CAPTIVATE (NCT02910583), a randomized phase II study, evaluates minimal residual disease (MRD)-guided treatment discontinuation following completion of first-line ibrutinib plus venetoclax treatment in patients with chronic lymphocytic leukemia (CLL). METHODS: Previously untreated CLL patients age < 70 years received three cycles of ibrutinib and then 12 cycles of combined ibrutinib plus venetoclax. Patients in the MRD cohort who met the stringent random assignment criteria for confirmed undetectable MRD (Confirmed uMRD) were randomly assigned 1:1 to double-blind placebo or ibrutinib; patients without Confirmed uMRD (uMRD Not Confirmed) were randomly assigned 1:1 to open-label ibrutinib or ibrutinib plus venetoclax. Primary end point was 1-year disease-free survival (DFS) rate with placebo versus ibrutinib in the Confirmed uMRD population. Secondary end points included response rates, uMRD, and safety. RESULTS: One hundred sixty-four patients initiated three cycles of ibrutinib lead-in. After 12 cycles of ibrutinib plus venetoclax, best uMRD response rates were 75% (peripheral blood) and 68% (bone marrow). Patients with Confirmed uMRD were randomly assigned to receive placebo (n = 43) or ibrutinib (n = 43); patients with uMRD Not Confirmed were randomly assigned to ibrutinib (n = 31) or ibrutinib plus venetoclax (n = 32). Median follow-up was 31.3 months. One-year DFS rate was not significantly different between placebo (95%) and ibrutinib (100%; arm difference: 4.7% [95% CI, -1.6 to 10.9]; P = .15) in the Confirmed uMRD population. After ibrutinib lead-in tumor debulking, 36 of 40 patients (90%) with high tumor lysis syndrome risk at baseline shifted to medium or low tumor lysis syndrome risk categories. Adverse events were most frequent during the first 6 months of ibrutinib plus venetoclax and generally decreased over time. CONCLUSION: The 1-year DFS rate of 95% in placebo-randomly assigned patients with Confirmed uMRD suggests the potential for fixed-duration treatment with this all-oral, once-daily, chemotherapy-free regimen in first-line CLL.

Phase 1 trial of ibrutinib and carfilzomib combination therapy for relapsed or relapsed and refractory multiple myeloma.

This phase 1, dose-finding study investigated ibrutinib and carfilzomib ± dexamethasone in patients with relapsed or relapsed/refractory multiple myeloma (≥2 lines of therapy including bortezomib and an immunomodulatory agent). Of 43 patients enrolled, 74% were refractory to bortezomib and 23% had high-risk cytogenetics. No dose-limiting toxicities were observed. The recommended phase 2 dose was ibrutinib 840 mg and carfilzomib 36 mg/m2 with dexamethasone. The most common ≥ grade 3 (>10%) treatment-emergent adverse events were hypertension, anemia, pneumonia, fatigue, diarrhea, and thrombocytopenia. Overall response rate was 67% (very good partial response, 21%; stringent complete response, 2%), with an additional 9% minimal response. Median progression-free survival was 7.2 months and was not inferior in refractory nor high-risk patients. Median overall survival was not reached. Ibrutinib plus carfilzomib demonstrated encouraging responses with a manageable safety profile in this advanced population.

PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers.

The spleen tyrosine kinase (SYK) regulates immune cell activation in response to engagement of a variety of receptors, making it an intriguing target for the treatment of inflammatory and autoimmune disorders as well as certain B-cell malignancies. We have previously reported on the discovery and preclinical characterization of PRT062607, a potent and highly selective inhibitor of SYK that exhibits robust anti-inflammatory activity in a variety of animal models. Here we present data from our first human studies aimed at characterizing the pharmacokinetics (PK), pharmacodynamics (PD), and safety of PRT062607 in healthy volunteers following single and multiple oral administrations. PRT062607 demonstrated a favorable PK profile and the ability to completely inhibit SYK activity in multiple whole-blood assays. The PD half-life in the more sensitive assays was approximately 24 hours and returned to predose levels by 72 hours. Selectivity for SYK was observed at all dose levels tested. Analysis of the PK/PD relationship indicated an IC50 of 324 nM for inhibition of B-cell antigen receptor-mediated B-cell activation and 205 nM for inhibition of FcεRI-mediated basophil degranulation. PRT062607 was safe and well tolerated across the entire range of doses. Clinical PK/PD was related to in vivo anti-inflammatory activity of PRT062607 in the rat collagen-induced arthritis model, which predicts that therapeutic concentrations may be safely achieved in humans for the treatment of autoimmune disease. PRT062607 has a desirable PK profile and is capable of safely, potently, and selectively suppressing SYK kinase function in humans following once-daily oral dosing.

The novel kinase inhibitor PRT062070 (Cerdulatinib) demonstrates efficacy in models of autoimmunity and B-cell cancer.

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate-mediated signaling or activation in B and T cells nor T-cell antigen receptor-mediated signaling in T cells, providing evidence for selectivity of action. Potent antitumor activity was observed in a subset of B-cell lymphoma cell lines. After oral dosing, PRT062070 suppressed inflammation and autoantibody generation in a rat collagen-induced arthritis model and blocked B-cell activation and splenomegaly in a mouse model of chronic B-cell antigen receptor stimulation. PRT062070 is currently under evaluation in a phase I dose escalation study in patients with B-cell leukemia and lymphoma (NCT01994382), with proof-of-concept studies in humans planned to assess therapeutic potential in autoimmune and malignant diseases.

The selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and function in vitro and in vivo and augments the activity of fludarabine in chronic lymphocytic leukemia.

B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.

Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 µM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 µM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 µM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Thienopyridines, but not elinogrel, result in off-target effects at the vessel wall that contribute to bleeding.

Clinical studies with clopidogrel or prasugrel show that although increased inhibition of P2Y(12) and platelet function improves efficacy, bleeding is also increased. Other preclinical and clinical studies have suggested a greater therapeutic index (TI) with reversible inhibitors and disproportionate effects of thienopyridines on bleeding at high doses. We used multiple in vivo (FeCl(3)-induced arterial thrombosis in mesenteric arteries, blood loss after tail transsection, and platelet deposition and wound closure time in a micropuncture model in mesenteric veins) and ex vivo (light transmittance aggregometry, prothrombin time, and activated partial thromboplastin time) mouse models to 1) compare the TI of clopidogrel, prasugrel, and elinogrel, a reversible, competitive antagonist, with that in P2Y(12)(-/-) mice and 2) determine whether the bleeding consequences of the thienopyridines are attributed only to the inhibition of P2Y(12). Data indicated greater (elinogrel) and decreased (thienopyridines) TI compared with that in P2Y(12)(-/-) mice. The impaired TI associated with the thienopyridines was not attributed to non-P2Y(12) activities on platelet function or coagulation but was related to a direct effect at the vessel wall (inhibition of vascular tone). Further analysis showed that the prasugrel off-target effect was dose- and time-dependent and of a reversible nature. In conclusion, the TI of thienopyridines in the mouse may be decreased by P2Y(12)-independent off-target effects at the vessel wall, whereas that of elinogrel may be enhanced by the reversible, competitive nature of the antiplatelet agent.

PRT-060318, a novel Syk inhibitor, prevents heparin-induced thrombocytopenia and thrombosis in a transgenic mouse model.

Heparin-induced thrombocytopenia (HIT) is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparin-platelet factor 4 complexes lead to FcγRIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT.

A porcine model for fixed drug eruptions in humans: the case of antipyrine in the Yucatan micropig.

To date, there is no acceptable animal model to investigate fixed drug eruptions (FDEs) in humans. We briefly report here observations suggesting that the Yucatan micropig may be a useful animal model for that purpose. During an investigation of antipyrine absorption and disposition, we observed the development of FDEs after intravenous administration of a 1 g dose. Our observations were consistent with those reported in several investigations of humans taking a single dose of antipyrine. To confirm these results, a naïve micropig was challenged. A male uncastrated Yucatan micropig (27.2 kg) was given a 1 g dose of antipyrine intravenously. After 30 days, this pig was rechallenged with the same intravenous dose of antipyrine (1 g). Blood samples were obtained to examine immunological endpoints. During the initial challenge, a fluid plaque (ca. 1-1.5 cm) appeared on the left hip of the pig ca. 6 h after dosing. After the rechallenge, inflamed pink patches were observed at the same sites where the blisters formed initially; however, no blisters re-formed. Changes of neutrophil, lymphocyte and eosinophil levels from baseline were noted 8 h after challenge. The micropig did not seem otherwise affected by the FDEs. These observations suggest that the Yucatan micropig, or swine in general, may be a useful animal model for detecting drugs that may cause FDEs in humans.

Protein expression pattern of P-glycoprotein along the gastrointestinal tract of the Yucatan micropig.

The purpose of this study is to characterize the distribution pattern of P-gp protein levels along the entire GI tract in the Yucatan micropig, which is being developed as a model for human drug bioavailability. Small and large intestines were freshly obtained and divided into about 37 segments and 10 segments, respectively (ca., 1 foot/segment). Epithelial cells from the small intestine were obtained by an elution method; whereas, a scraping method was applied to the large intestine. Total cellular protein was isolated from the epithelial cells. Western blot analysis using P-gp antibody showed that the amount of P-gp protein increased distally from the duodenum to the ileum over approximately a 10-fold range. P-gp protein in the large intestine was present at a higher level in the central portion, but the absolute amount was much less than what was found in the small intestine.

Sensitive and rapid isocratic liquid chromatography method for the quantitation of curcumin in plasma.

An HPLC assay was developed using three methods of plasma sample preparation in order to quantitate curcumin, the main constituent in the herbal dietary supplement turmeric. Each method involves simple and rapid processing of samples (either an ethyl acetate or chloroform extraction) with resulting different quantitation limits for curcumin. The assay was developed in an effort to quantify extremely low curcumin plasma concentrations observed in preliminary in vivo studies. The most sensitive assay can reliably detect concentrations down to 2.5 ng/ml. Plasma quantitation was precise and accurate based on both intra- and inter-day validations as indicated by low values for coefficients of variation and bias, respectively (< or =15%). The analytical validation was reproducible between different analysts. The resulting analytical method couples desired sensitivity with the ease of an isocratic system.